Prokaryotic genes differ from eukaryotic genes in that every base pair in a prokaryotic gene is reflected in the mRNA base sequence. In eukaryotic genes there are often intervening sequences (introns) that do not appear in the mRNA base sequence for the gene product. The DNA sequences that are expressed and retained in the final mRNA product are “exons”.
The entire DNA sequence, including exons and introns, are transcribed to produce a precursor hnRNA of the mature mRNA. However, during the splicing of pre-mRNA the introns are excised out and the exons are spliced together via two-step trans-esterification reactions carried out by the spliceosome, which consists of five ribonucleoprotein particles (RNPs). Genes from eukaryotic organisms contain a variable number of introns of varying sizes. For example, the gene for mouse Tbc1d2 gene encoding TBC1 domain family, member 2 contains 12 introns, the mouse Col1a1 gene coding for procollagen, type I, alpha 1, contains 50 introns.
The excision or splicing of spliceosomal introns occurs in the cell nucleus and is mediated by splicosome which consists of five ribonucleoprotein particles (RNPs). The splicosomal intron comprises a 5′ splice site, 3′ splice site and branch site. The spliceosomal small nuclear RNAs have highly conserved secondary structures similar to domains of self-splicing group II introns which are thought to be ancestors of the spliceosomal introns.
Ever since their discovery about 30 years ago, introns have intrigued the scientific community and stimulated debate about the nature and timing of their origin. There has also been curiosity about the apparent recent explosion in intron number in mammals and its contribution to expanded protein diversity and regulation through alternative splicing pathways. Correct removal of introns from genes has become a central issue in the medical research field. Otherwise, it leads to various human diseases such as cancers, autosomal recessive disorder, spinal muscular atrophy. However there are no methods to accurately identify the introns, that is, to accurately define exon/intron boundaries. We recently reported that many newly-acquired introns in the human genome share a signature of identical 5′ and 3′ splicing junctions consistent with an origin via segmental DNA duplication. It makes possible to accurately predict and annotation of mammalian genes and opens up many possibilities to identify novel control trans- or cis-elements and to predict novel alternatively spliced mRNA isoforms.
The specific markers are 20 bp upstream (E5) and 20 bp downstream (I5) nucleotides of 5′ splicing sites and 20 bp upstream (I3) and 20 bp downstream (E3) nucleotides of 3′ splicing sites.
Therefore it is an objective of the present invention to provide a method for determining the presence of introns in eukaroytic genome.
The invention also provides for diagnostic methods employing the characteristic markers of associated introns and exons.
Another objective of the present invention is to provide a method for identification of novel cis- or trans-elements which accurately guide pre-mRNA and alternative splicing.
It is also an objective of the present invention to provide a method for detecting novel introns and exons, therefore, novel mRNA isoforms or protein isoforms.
Yet another objective of the present invention is to provide a method for predicting genes associated with various diseases or genetic mutation based on the splicing junction profile of a given sample of genetic material.
Another objective of the present invention is to provide a method which may be utilized to design DNA/RNA oligomers to detect, identify and screen genetic variations or novel isoforms (DNA, RNA and/or proteins).
It is a further objective of the present invention to provide research and diagnostic methodologies that employ standard laboratory equipment, are relatively inexpensive to perform and which do not require extensive operator skill or training.
Another objective is to provide methods for identifying trans-elements which accurately guide pre-mRNA and alternative splicing.
Various other objectives and advantages of the present invention will become apparent to those skilled in the art as a more detailed description is set forth below.